Prakasit Rattanatanyong

LINE-1 methylation in the peripheral blood mononuclear cells of cancer patients.

BACKGROUND Recently, we classified LINE-1 loci according to their methylation statuses and found that the percentage of hypomethylated LINE-1 loci ((u)C(u)C) can differentiate between the peripheral blood mononuclear cells (PBMCs) of oral cancer patients and normal controls with a higher specificity and sensitivity than overall methylation levels. Here, we evaluated the LINE-1 methylation levels and patterns in PBMCs from patients with cancers of the nasopharynx, lung, liver, bile duct, breast and colon. METHODS Combined Bisulfite Restriction Analysis (COBRA) of LINE-1 loci was performed to examine the LINE-1 methylation statuses of PBMCs from 216 cancer patients with 6 different types of cancer compared with 144 normal controls. RESULTS Only colorectal and nasopharyngeal cancer samples were found to have lower levels of overall LINE-1 methylation compared with normal controls (p<0.0001 and p=0.0022). However, %(u)C(u)C in cancers of the colon, liver, lung and nasopharynx was significantly higher compared with normal controls (p<0.0001, p<0.0001, p=0.01 and p=0.001, respectively). Furthermore, ROC curve analyses of these four cancer types also demonstrated the potential of %(u)C(u)C as a biomarker for cancer diagnosis. CONCLUSION Changes in the levels and patterns of genome-wide methylation of PBMCs are associated with cancer risk. For LINE-1, %(u)C(u)C is a more effective tumour marker for determining cancer risk than overall methylation levels.

Replication independent DNA double-strand break retention may prevent genomic instability

BackgroundGlobal hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability.ResultsWe observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51.ConclusionsMethylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.

Differences in LINE-1 methylation between endometriotic ovarian cyst and endometriosis-associated ovarian cancer.

Background Endometriosis in endometriosis-associated ovarian cancer (EAOC) refers to lesions that can derive from endometriotic ovarian cysts (ECs) that form in the ovarian endometrium with the potential to transform into full-blown ovarian cancer. Hypomethylation of long interspersed element-1 (LINE-1 or L1) is a common epigenomic event in several cancers and is strongly associated with ovarian cancer progression. Objectives To evaluated alterations in LINE-1 methylation between EC, ovarian endometrioid adenocarcinoma (OEA), EAOC, and ovarian clear cell carcinoma (OCC). Methods/ Materials First, LINE-1 methylation status in 19 normal endometrium, 29 EC, 35 OCC, and 22 OEA tissues from unrelated samples were compared. Then, specific areas of eutopic endometrium, contiguous endometriosis, and cancer arising from 16 EAOCs were collected by microdissection and analyzed for LINE-1 methylation status. Results The total LINE-1 methylation levels were significantly different among the endometrium, endometriosis, and ovarian cancer (P < 0.001). A stepwise decrease in LINE-1 methylation was observed in the following order: normal endometrium, EC, OEA, and OCC. Interestingly, endometriosis in EAOC of both OEA (P = 0.016) and OCC (P = 0.003) possessed a higher percentage of LINE-1 unmethylated loci than EC. Conclusion Our data implicate that LINE-1 hypomethylation is an early molecular event involved in OEA and OCC malignant transformation. Precise measurements of LINE-1 methylation may help to distinguish EC and endometriosis in EAOC.

LINE-1 and Alu methylation patterns in lymph node metastases of head and neck cancers.

BACKGROUND The potential use of hypomethylation of Long INterspersed Element 1 (LINE-1) and Alu elements (Alu) as a biomarker has been comprehensively assessed in several cancers, including head and neck squamous cell carcinoma (HNSCC). Failure to detect occult metastatic head and neck tumors on radical neck lymph node dissection can affect the therapeutic measures taken. OBJECTIVE The aim of this study was to investigate the LINE-1 and Alu methylation status and determine whether it can be applied for detection of occult metastatic tumors in HNSCC cases. METHODS We used the Combine Bisulfite Restriction Analysis (COBRA) technique to analyse LINE-1 and Alu methylation status. In addition to the methylation level, LINE-1 and Alu loci were classified based on the methylation statuses of two CpG dinucleotides in each allele as follows: hypermethylation (mCmC), hypomethylation (uCuC), and 2 forms of partial methylation (mCuC and uCmC). Sixty-one lymph nodes were divided into 3 groups: 1) non-metastatic head and neck cancer (NM), 2) histologically negative for tumor cells of cases with metastatic head and neck cancer (LN), and 3) histologically positive for tumor cells (LP). RESULTS Alu methylation change was not significant. However, LINE-1 methylation of both LN and LP was altered, as demonstrated by the lower LINE-1 methylation levels (p<0.001), higher percentage of mCuC (p<0.01), lower percentage of uCmC (p<0.001) and higher percentage of uCuC (p<0.001). Using receiver operating characteristic (ROC) curve analysis, %uCmC and %mCuC values revealed a high level of AUC at 0.806 and 0.716, respectively, in distinguishing LN from NM. CONCLUSION The LINE-1 methylation changes in LN have the same pattern as that in LP. This epigenomic change may be due to the presence of occult metastatic tumor in LN cases.

Replication-Independent Endogenous DNA Double-Strand Breaks in Saccharomyces cerevisiae Model

Without exposure to any DNA-damaging agents, non-dividing eukaryotic cells carry endogenous DNA double-strand breaks (EDSBs), or Replication-Independent (RIND)-EDSBs. In human cells, RIND-EDSBs are enriched in the methylated heterochromatic areas of the genome and are repaired by an ATM-dependent non-homologous end-joining pathway (NHEJ). Here, we showed that Saccharomyces cerevisiae similarly possess RIND-EDSBs. Various levels of EDSBs were detected during different phases of the cell cycle, including G0. Using a collection of mutant yeast strains, we investigated various DNA metabolic and DNA repair pathways that might be involved in the maintenance of RIND-EDSB levels. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in NHEJ DNA repair and in suppression of heterochromatin formation. RIND-EDSB levels were also upregulated when genes encoding histone deacetylase, endonucleases, topoisomerase, and DNA repair regulators were deleted. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Likewise, RIND-EDSB levels were also decreased in human cells lacking HMGB1. Therefore, we conclude that the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems.

Maternal uniparental disomy of chromosome 16 resulting in hemoglobin Bart's hydrops fetalis.

To the Editor: Since the first cases of maternal uniparental disomy for chromosome 16 or UPD(16)mat was described by Kalousek et al. in 1993, a handful of cases have been diagnosed in newborn infants and terminated fetuses (1–4). Most cases are associated with confined placental mosaicism, with high incidence of adverse pregnancy outcome (4, 5). The most common findings include intrauterine growth retardation, single umbilical artery, imperforate anus, and pulmonary and cardiac malformations (1, 2, 4, 6, 7). Also, UPD(16)mat individual with normal phenotypes has been reported (8). The only confirmed case with UPD(16)pat was isodisomic and identified through pre-natal detection for abnormal maternal serum screening; eventually, the child was quite normal (9). Herein, we describe a hemoglobin (Hb) Bart’s hydroptic fetus and fetal malformations caused by UPD(16)mat. The counselee is a 34-year-old G1P0 pregnant Thai woman affected with Hb H disease caused by heterozygous Southeast Asian (–SEA) and the 3.7-kb deletions of the alpha1 and 2 globin genes, respectively. Her husband is Hb E heterozygote. Ultrasound performed at a gestation of 23 weeks showed a hydroptic fetus. Fetal blood from cordocentesis showed Hb Bart’s hydrops fetalis with –SEA globin1 gene deletion but not the other types of alpha1 globin gene mutations, suggesting the following possibilities: non-paternity, another rare but as yet unidentified alphathalassemia 1 mutation inherited from the father, or UPD(16)mat. Fetal autopsy showed characteristic features of Hb Bart’s hydrops including interstitial edema of skin, cardiomegaly, and hepatosplenomegaly with extramedullary hemopoiesis. Single umbilical artery, bilobed right lung and ileal Meckel’s diverticulum were found. Retrospectively, a two-vessel cord was noted on pre-natal ultrasonogram. Peripheral blood culture for fetal karyotype analysis was unsuccessful. It is unlikely that the husband has an unidentified alpha-thalassemia 1 gene deletion, given normal hematocrit, mildly small red cell size, and percent Hb E as expected in a heterozygous Hb E individual. Normally, heterozygote for Hb E with co-inheritance of alpha-thalassemia 1 will

LINE-1 Methylation Patterns as a Predictor of Postmolar Gestational Trophoblastic Neoplasia

Objective. To study the potential of long interspersed element-1 (LINE-1) methylation change in the prediction of postmolar gestational trophoblastic neoplasia (GTN). Methods. The LINE-1 methylation pattern from first trimester placenta, hydatidiform mole, and malignant trophoblast specimens were compared. Then, hydatidiform mole patients from 11999 to 2010 were classified into the following 2 groups: a remission group and a group that developed postmolar GTN. Specimens were prepared for a methylation study. The methylation levels and percentages of LINE-1 loci were evaluated for their sensitivity, specificity, and accuracy for the prediction of postmolar GTN. Results. First, 12 placentas, 38 moles, and 19 malignant trophoblast specimens were compared. The hydatidiform mole group had the highest LINE-1 methylation level (p = 0.003) and the uCuC of LINE-1 increased in the malignant trophoblast group (p ≤ 0.001). One hundred forty-five hydatidiform mole patients were classified as 103 remission and 42 postmolar GTN patients. The %mCuC and %uCmC of LINE-1 showed the lowest p value for distinguishing between the two groups (p < 0.001). The combination of the pretreatment β-hCG level (≥100,000 mIU/mL) with the %mCuC and %uCmC, sensitivity, specificity, PPV, NPV, and accuracy modified the levels to 60.0%, 92.2%, 77.4%, 83.8%, and 82.3%, respectively. Conclusions. A reduction in the partial methylation of LINE-1 occurs early before the clinical appearance of malignant transformation. The %mCuC and %uCmC of LINE-1s may be promising markers for monitoring hydatidiform moles before progression to GTN.

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

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.

Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings.

Alu elements and long interspersed element-1 (LINE-1 or L1) are two major human intersperse repetitive sequences. Lower Alu methylation, but not LINE-1, has been observed in blood cells of people in old age, and in menopausal women having lower bone mass and osteoporosis. Nevertheless, Alu methylation levels also vary among young individuals. Here, we explored phenotypes at birth that are associated with Alu methylation levels in young people. In 2010, 249 twenty-years-old volunteers whose mothers had participated in a study association between birth weight (BW) and nutrition during pregnancy in 1990, were invited to take part in our present study. In this study, the LINE-1 and Alu methylation levels and patterns were measured in peripheral mononuclear cells and correlated with various nutritional parameters during intrauterine and postnatal period of offspring. This included the amount of maternal intake during pregnancy, the mother’s weight gain during pregnancy, birth weight, birth length, and the rate of weight gain in the first year of life. Catch-up growth (CUG) was defined when weight during the first year was >0.67 of the standard score, according to WHO data. No association with LINE-1 methylation was identified. The mean level of Alu methylation in the CUG group was significantly higher than those non-CUG (39.61% and 33.66 % respectively, P < 0.0001). The positive correlation between the history of CUG in the first year and higher Alu methylation indicates the role of Alu methylation, not only in aging cells, but also in the human growth process. Moreover, here is the first study that demonstrated the association between a phenotype during the newborn period and intersperse repetitive sequences methylation during young adulthood.

Alu Methylation in Serum from Patients with Nasopharyngeal Carcinoma

BACKGROUND Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. Alu elements are among the most prevalent repetitive sequences and constitute 11% of the human genome. Although Alu methylation has been evaluated in many types of cancer, few studies have examined the levels of this modification in serum from NPC patients. OBJECTIVE To compare the Alu methylation levels and patterns between serum from NPC patients and normal controls. MATERIALS AND METHODS Sera from 50 NPC patients and 140 controls were examined. Quantitative combined bisulfite restriction analysis-Alu (qCOBRA-Alu) was applied to measure Alu methylation levels and characterize Alu methylation patterns. Amplified products were classified into four patterns according to the methylation status of 2 CpG sites: hypermethylated (methylation at both loci), partially methylated (methylation of either of the two loci), and hypomethylated (unmethylated at both loci). RESULTS A comparison of normal control sera with NPC sera revealed that the latter presented a significantly lower methylation level (p=0.0002) and a significantly higher percentage of hypomethylated loci (p=0.0002). The sensitivity of the higher percentage of Alu hypomethyted loci for distinguishing NPC patients from normal controls was 96%. CONCLUSIONS Alu elements in the circulating DNA of NPC patients are hypomethylated. Moreover, Alu hypomethylated loci may represent a potential biomarker for NPC screening.