Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.</p><p><b>METHODS</b>A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone.</p><p><b>RESULTS</b>The LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05).</p><p><b>CONCLUSIONS</b>LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.</p>

Epigenetic modification of long interspersed elements-1 in cumulus cells of mature and immature oocytes from patients with polycystic ovary syndrome / 대한생식의학회지

OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

Auditory Neuropathy/Dyssynchrony: A Retrospective Analysis of 15 Cases

Introduction Auditory neuropathy/dyssynchrony (AN/AD) comprises a spectrum of pathology affecting the auditory pathways anywhere from the inner hair cells to the brainstem. It is characterized by an absent or atypical auditory brainstem response (ABR) with preservation of the cochlear microphonics and/or otoacoustic emissions (OAEs). Objective Retrospective analysis of patients with AN/AD. Methods Fifteen patients with AN/AD were included in this study and their records were retrospectively investigated. Results Possible etiology of AN/AD was neonatal hyperbilirubinemia in three patients, family history of hearing loss in three patients, consanguineous marriage in two patients, head trauma in two patients, mental motor retardation in one patient, cerebrovascular disease in one patient, and there was no apparent cause in three patients. Conclusion Otolaryngologists should keep in mind the diagnosis of AN/AD especially in patients complaining of difficulty in hearing and speech and audiological evidence of disassociation between pure tone and speech audiometry. ABR and OAE testing is recommended in these patients for AN/AD diagnosis. .

Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>DNA hypomethylation of long interspersed nuclear elements-1 (LINEs-1) occurs during carcinogenesis, whereas information addressing LINE-1 methylation in Wilms tumor (WT) is limited. The main purpose of our study was to quantify LINE-1 methylation levels and evaluate their relationship with relative telomere length (TL) in WT.</p><p><b>METHODS</b>We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction (PCR) pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line. Significant changes were analyzed by t-tests.</p><p><b>RESULTS</b>LINE-1 methylation levels were significantly lower (P < 0.05) and relative TLs were significantly shorter (P < 0.05) in WT compared with normal kidney. There was a significant positive relationship between LINE-1 methylation and relative TL in WT (r = 0.671, P = 0.001). LINE-1 Methylation levels were significantly associated with global DNA methylation (r = 0.332, P < 0.01). In addition, relative TL was shortened and LINE-1 methylation was decreased in a WT cell line treated with the hypomethylating agent 5-aza-2'-deoxycytidine compared with untreated WT cell line.</p><p><b>CONCLUSION</b>These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT.</p>

Hipometilação do LINE-1 está Associada ao Risco de Doença Cardíaca Coronariana na População Chinesa / LINE-1 Hypomethylation is Associated with the Risk of Coronary Heart Disease in Chinese Population

Fundamentos: O nível de metilação global do ADN de leucócitos no sangue tem sido associado ao risco de doença arterial coronariana (DAC), com resultados inconsistentes em diferentes populações. Faltam dados semelhantes da população chinesa, onde diferentes fatores genéticos, de estilo de vida e ambientais podem afetar a metilação do ADN e sua relação com o risco de DCC. Objetivos: Analisar se a metilação global está associada ao risco de doença coronariana na população chinesa. Métodos: Foram incluídos um total de 334 casos de DCC e 788 controles saudáveis. A metilação global do ADN de leucócitos de sangue foi estimada por meio da análise das repetições do LINE-1 usando pirosequenciamento de bissulfito. Resultados: Em uma análise inicial restrita aos controles o nível do LINE-1 diminui significativamente com a idade avançada, colesterol total elevado, e diagnóstico de diabetes. Na análise de caso-controle, a redução da metilação do LINE-1 foi associada ao aumento do risco de DCC, tendo a análise por quartil revelado uma odds ratio (IC 95%) de 0,9 (0,6-1,4), 1,9 (1,3-2,9) e 2,3 (1,6 3.5) para o terceiro, segundo e primeiro (o mais baixo) quartil (P da tendência < 0,001), respectivamente, em comparação com o quarto (o mais alto) quartil. A metilação inferior (< mediana) do LINE-1 esteve associada a 2,2 vezes (IC 95% = 1,7-3,0) o aumento de risco de doença coronariana. As estimativas de risco de DCC menores relacionadas com o LINE-1 tenderam a ser mais fortes entre os indivíduos com maior tercil de homocisteína (P interação = 0,042) e naqueles com diagnóstico de hipertensão arterial (P interação = 0,012). Conclusão: A hipometilação do LINE-1 está ...

Background: Global methylation level in blood leukocyte DNA has been associated with the risk of coronary heart disease (CHD), with inconsistent results in various populations. Similar data are lacking in Chinese population where different genetic, lifestyle and environmental factors may affect DNA methylation and its risk relationship with CHD. Objectives: To examine whether global methylation is associated with the risk of CHD in Chinese population. Methods: A total of 334 cases with CHD and 788 healthy controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing LINE-1 repeats using bisulfite pyrosequencing. Results: In an initial analysis restricted to control subjects, LINE-1 level reduced significantly with aging, elevated total cholesterol, and diagnosis of diabetes. In the case-control analysis, reduced LINE-1 methylation was associated with increased risk of CHD; analysis by quartile revealed odds ratios (95%CI) of 0.9 (0.6-1.4), 1.9 (1.3-2.9) and 2.3 (1.6-3.5) for the third, second and first (lowest) quartile (Ptrend < 0.001), respectively, compared to the fourth (highest) quartile. Lower (<median) LINE-1 methylation was associated with a 2.2-fold (95%CI = 1.7-3.0) increased risk of CHD. The lower LINE-1-related CHD risk estimates tended to be stronger among subjects with the highest tertile of homocysteine (Pinteraction = 0.042) and those with diagnosis of hypertension (Pinteraction = 0.012). Conclusion: LINE-1 hypomethylation is associated with the risk of CHD in Chinese population. Potential CHD risk factors such as older age, elevated total cholesterol, and diagnosis of diabetes may have impact on global DNA methylation, whereby exerting their effect on CHD risk. .

Relationship between Line 1 methylation and clinical data of non-small cell lung cancer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the impact of Line-1 methylation on clinical features of non-small cell lung cancer and its connection with smoking and other living habits.</p><p><b>METHODS</b>Pyrosequencing was used to determine the extent of Line-1 methylation in cancer and adjacent tissues derived from 197 patients with primary non-small cell lung cancer. Non-conditional logistic regression analysis was performed to correlate the level of Line-1 methylation with clinical features and living habits of the patients.</p><p><b>RESULTS</b>Line-1 methylation for cancer tissue and adjacent tissue has measured 68.20±11.63 and 78.90±2.09, respectively (P < 0.01), and has been associated with TNM staging, smoking history and histopathological types.</p><p><b>CONCLUSION</b>Lung cancer tissue Line-1 methylation level is closely related with clinical features and smoking. There is also a correlation between histopathological types of lung cancer and relative hypomethylation of Line-1.</p>

A novel deletion cluster at 13q14.2-q21.33 in an 80-year man with late onset leukemia: Clinical and molecular findings.

Chromosomal deletions are among the most common genetic events observed in hematologic malignancies; loss of genetic material is regarded as a hallmark of putative tumor suppressor gene localization. We have identified an unusual cluster of deletions at 13q14.2-13q21.33 in an 80-year-old father of a monozygotic twin pair discordant for schizophrenia, who developed chronic leukemia (CLL) at age 69. MATERIALS AND METHODS: The breakpoints for individual deletions in this cluster was identified by Affymetrix Human Array 6.0 screening. RESULTS: The deleted segments harbours a number of genes, most associated with cancer as well as a high concentration of LINEs, SINEs and related repeats. The derived chromosome represents an intra-chromosomal re-arrangement that quickly overtook blood progenitor cells probably before age 69 as a cause of CLL. CONCLUSIONS: The study highlights the role of ongoing de novo changes at susceptible sites, such as repeat rich regions, in the human genome. Also, it argues for the involvement of genes/deletions in the 13q(14.2-21.33) region in the development of CCL.

Transposable Elements: No More 'Junk DNA'

Since the advent of whole-genome sequencing, transposable elements (TEs), just thought to be 'junk' DNA, have been noticed because of their numerous copies in various eukaryotic genomes. Many studies about TEs have been conducted to discover their functions in their host genomes. Based on the results of those studies, it has been generally accepted that they have a function to cause genomic and genetic variations. However, their infinite functions are not fully elucidated. Through various mechanisms, including de novo TE insertions, TE insertion-mediated deletions, and recombination events, they manipulate their host genomes. In this review, we focus on Alu, L1, human endogenous retrovirus, and short interspersed element/variable number of tandem repeats/Alu (SVA) elements and discuss how they have affected primate genomes, especially the human and chimpanzee genomes, since their divergence.

Human LINE1 endonuclease domain as a putative target of SARS-associated autoantibodies involved in the pathogenesis of severe acute respiratory syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Severe acute respiratory syndrome (SARS) is a disease with a mortality of 9.56%. Although SARS is etiologically linked to a new coronavirus (SARS-CoV) and functional cell receptor has been identified, the pathogenesis of the virus infection is largely unclear.</p><p><b>METHODS</b>The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house. Further investigations of target antigen included reviews of phage display technique, rapid amplification of cDNA ends (RACE) technique, protein expression and purification, Western blotting validation, serological and immunohistochemical staining in postmortem tissue.</p><p><b>RESULTS</b>A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease. Human long interspersed nuclear element 1 (LINE1) gene endonuclease (EN) domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients. Anti-EN antibody was positive in the sera of 40.9% of SARS patients.</p><p><b>CONCLUSIONS</b>Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies, which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.</p>

Chromosomal Losses are Associated with Hypomethylation of the Gene-Control Regions in the Stomach with a Low Number of Active Genes

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Alu and L1 Retroelements Are Correlated with the Tissue Extent and Peak Rate of Gene Expression, Respectively

We exploited the serial analysis of gene expression (SAGE) libraries and human genome database in silico to correlate the breadth of expression (BOE; housekeep-ing versus tissue-specific genes) and peak rate of expression (PRE; high versus low expressed genes) with the density distribution of the retroelements. The BOE status is linearly associated with the density of the sense Alus along the 100 kb nucleotides region upstream of a gene, whereas the PRE status is inversely correlated with the density of antisense L1s within a gene and in the up- and downstream regions of the 0-10 kb nucleotides. The radial distance of intranuclear position, which is known to serve as the global domain for transcription regulation, is reciprocally correlated with the fractions of Alu (toward the nuclear center) and L1 (toward the nuclear edge) elements in each chromosome. We propose that the BOE and PRE statuses are related to the reciprocal distribution of Alu and L1 elements that formulate local and global expression domains.

Differential organization of a LINE-1 family in Indian pygmy field mice.

Southern blot hybridization analysis of genomic DNAs digested with restriction endonuclease EcoR I and Ava II from Mus musculus domesticus, Mus booduga and Mus terricolor with a cloned repetitive DNA fragment of Mus booduga as a probe showed difference in restriction pattern of this DNA in these three species. Further Southern analysis of the BamH I digested genomic DNAs from these species hybridized with cloned DNA fragment as a probe and sequencing of the cloned DNA revealed that this 252 bp cloned DNA fragment is a part of BamHI repeat element of genus Mus and is 87% homologous to the contiguous portion of the Mus musculus domesticus LINE-1 element. The species specific fragment pattern generated by different restriction endonucleases using this DNA as a probe revealed difference in the organization of LINE-1 repetitive element in the three species of genus Mus.

Sequence analysis of canine LINE-1 elements and p53 gene in canine transmissible venereal tumor

LINEs (long interspersed nuclear elements or long interspersed repeated DNA elements) contains two open reading frames (ORFs), ORF1 and ORF2. We analysed the ORF2 located in the 5' region to the first exon of oncogene c-myc in canine transmissible venereal tumor (TVT) cell. We also showed the transcription activation was induced by this TVT-LINE sequence using CAT assay. To identify the mutation of tumor suppressor gene, sequence analysis of p53 from TVT cell was performed. We identified the point mutation of 964 nucleotide (T-->C) resulting in the change of amino acid (Phe-->Ser) of p53 tumor suppressor protein.

The full sequence of intron 51 of dystrophin gene and its characteristic of sequence / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To finish the work of sequencing the full sequence of intron 51 of dystrophin gene and understand its characteristic of sequence.</p><p><b>METHODS</b>The whole intron 51 was sequenced by primer walking. The sequencing results were analyzed by repeat sequences, matrix attachment region (MAR) and topoisomerase II cleavage sites. The residue sequences, after removal of the repetitive sequences, were subjected to the analysis of CpG islands, promoter, open reading frame (ORF) and unidentified low copy repeat sequence.</p><p><b>RESULTS</b>The acquired intron 51 sequence was composed of 38725 bp. Repetitive sequences constituted 37.53% of total intron sequence. The overall G+C content of intron 51 was 36.34%. There are four potential MARs in intron 51. Three of them are clustered in the 12 kb region near exon 51. Numerous ORFs were found on both strands, but no homologues proteins were found in Genbank CDS transcriptional peptide, PDB, SwissProt, PIR and PRF databases.</p><p><b>CONCLUSION</b>The expansion of intron 7 over the last 120 million years was mainly the result of L1 insertion into intron 7, and not all of repetitive sequences are associated with chromosomal rearrangement. No sequence of functional significance was found in intron 51. The results suggest that the cluster of MARs may be associated with the instability of intron 51.</p>

Elementos Transponíveis e Doenças Humanas: da Instabilidade Genônica à Carcinogênese / Transposable Elements and Human Diseases: from Genomic Instability to Carcinogenesis

Os elementos transponíveis säo sequências de DNA que podem se mover no genoma e por isso säo a principal fonte de mutações espontâneas. Dez a 15 por cento do DNA humano säo compostos pelos transposons Alu e Line-1. O grande número desses elementos, no genoma, propicia ampla oportunidade para recombinaçäo homóloga desigual, ocasionando deleções ou duplicações gênicas e mesmo alterações mais complexas do material genético. Säo citados na literatura diversos casos de doenças causadas pela inserçäo de Alu e Line-1 em diversos genes, tanto na linhagem germinativa como somática. Também tem sido encontrada uma correlaçäo direta entre a hipometilaçäo de sequências regulatórias de Line-1 e a presença de produtos de transcriçäo, ou de proteína por ele produzida, em teratocarcinomas, tumores da linhagem germinativa e em alguns cânceres de cérebro e mama. Tal correlaçäo sugere que esses elementos estäo ativos e podem estar envolvidos na iniciaçäo e na manutençäo do estado maligno, principalmente porque a desmetilaçäo do DNA genômico parece ser um evento precoce na progressäo tumoral