Quantitative Fluorescence In Situ Hybridization Research Articles

Leukocyte telomere length positively correlates with duration of lithium treatment in bipolar disorder patients

Bipolar disorder (BD) has been suggested to be associated with accelerated aging and premature cell senescence. While findings on shorter telomeres in BD are controversial, a recent study showed that long-term lithium treatment correlates with longer telomeres in BD. In our study, we sought to investigate the correlation between leukocyte telomere length (LTL) and long-term lithium treatment in a sample of 200 BD patients characterized for lithium response. We also compared data from two different methods commonly used to measure telomere length, quantitative PCR (qPCR) and quantitative fluorescence in situ hybridization (Q-FISH). We also measured, for the first time, the effect of lithium in vitro on the expression of the telomerase gene in human-derived neural progenitor cells (NPCs). Our findings showed that LTL correlated negatively with age (p=0.0002) and was independent of sex, diagnosis, age at onset, suicidal behavior, number of mood episodes, response to lithium and use of other psychotropic medications. After correcting for age, LTL was positively correlated with lithium treatment duration in patients treated for more than two years (n=150, R=0.17, p=0.037). There was a significant correlation between data measured with qPCR and Q-FISH (p=0.012, R=0.826). Lithium treatment increased telomerase expression in NPCs, though this effect was not statistically significant.Our data support previous findings showing that long-term lithium treatment associates with longer telomeres in BD, though this effect appeared to be independent from clinical response to the treatment. Moreover, we suggested for the first time that lithium increases the expression of telomerase gene in human neural progenitor cells.

Determination of True ERBB2 Gene Amplification in Breast Cancer by Quantitative PCR Using a Reference and a Novel Control Gene.

Human epidermal growth factor receptor 2 (ERBB2/HER2) is amplified and overexpressed in 20% to 25% of breast carcinomas, correlates with poor outcome, and is an indication for treatment with trastuzumab. Accurate assessment of ERBB2 status is crucial for proper prognosis and to offer appropriate treatment for patients. ERBB2 status is generally determined by immunohistochemistry or fluorescence in situ hybridization (FISH), and sporadically by quantitative real-time polymerase chain reaction (PCR). We developed a new algorithm, termed quantitative PCR algorithm (QPA) score, and compared its performance with the gold standard FISH assay. The QPA is a computation of the relative number of copies of the ERBB2 gene with respect to a nonstandard, short-arm centromeric sequence on chromosome 17, and referenced to a single-copy gene, RPP30. This provides a more reliable determination of ERBB2 amplification, reducing the false polysomy 17 error. A total of 69 breast carcinoma samples were tested for quantitative real-time PCR and FISH, and the degree of concordance was analyzed. Sixty-two cases were in agreement between the 2 methods, and the contingency study assigned a κ value of 0.729 for their correlation. A receiver operating characteristic analysis was used to determine the optimal cut-off point for ERBB2 amplification, which was estimated at a QPA=1.53 (sensitivity=0.863; specificity=0.944). Our data conclude that the QPA is able to determine ERBB2 gene status with high accuracy, while also overcoming the limitations of conventional techniques and providing better cost-effectiveness.

Urinary Biomarkers for Diagnosis of Bladder Cancer: A Systematic Review and Meta-analysis.

Urinary biomarkers may be a useful alternative or adjunct to cystoscopy for diagnosis of bladder cancer. To systematically review the evidence on the accuracy of urinary biomarkers for diagnosis of bladder cancer in adults who have signs or symptoms of the disease or are undergoing surveillance for recurrent disease. Ovid MEDLINE (January 1990 through June 2015), Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and reference lists. 57 studies that evaluated the diagnostic accuracy of quantitative or qualitative nuclear matrix protein 22 (NMP22), qualitative or quantitative bladder tumor antigen (BTA), fluorescence in situ hybridization (FISH), fluorescent immunohistochemistry (ImmunoCyt [Scimedx]), and Cxbladder (Pacific Edge Diagnostics USA) using cystoscopy and histopathology as the reference standard met inclusion criteria. Case-control studies were excluded. Dual extraction and quality assessment of individual studies. Overall strength of evidence (SOE) was also assessed. Across biomarkers, sensitivities ranged from 0.57 to 0.82 and specificities ranged from 0.74 to 0.88. Positive likelihood ratios ranged from 2.52 to 5.53, and negative likelihood ratios ranged from 0.21 to 0.48 (moderate SOE for quantitative NMP22, qualitative BTA, FISH, and ImmunoCyt; low SOE for others). For some biomarkers, sensitivity was higher for initial diagnosis of bladder cancer than for diagnosis of recurrence. Sensitivity increased with higher tumor stage or grade. Studies that directly compared the accuracy of quantitative NMP22 and qualitative BTA found no differences in diagnostic accuracy (moderate SOE); head-to-head studies of other biomarkers were limited. Urinary biomarkers plus cytologic evaluation were more sensitive than biomarkers alone but missed about 10% of bladder cancer cases. Restricted to English-language studies; no search for studies published only as abstracts; statistical heterogeneity present in most analyses; few studies for qualitative NMP22, quantitative BTA, and Cxbladder; and methodological shortcomings in almost all studies. Urinary biomarkers miss a substantial proportion of patients with bladder cancer and are subject to false-positive results in others. Accuracy is poor for low-stage and low-grade tumors. Agency for Healthcare Research and Quality. (PROSPERO registration number: CRD42014013284).

Three Steps to the Diagnosis of Adult Ph-like ALL

Introduction: Philadelphia chromosome-likeacute lymphoblasic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to BCR-ABL-positive ALL and is associated with particularly poor prognosis. Aberrant gene-expression, point mutations or fusion translocations cause activation of either the ABL1 or JAK signaling pathways. The diagnosis of Ph-like ALL is challenging but of vital importance as it is likely to respond to targeted therapy with ABL or JAK inhibitors.Aims: To develop a robust and efficient stepwise diagnostic work-up to identify Ph-like ALL in routine diagnostics.Patients and Methods: We studied 132 adult precursor B-ALL patients (pts) lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 (age 18-85 years, median 54 years). Precursor B-ALL was diagnosed by immunophenotyping in all cases. The following methods were performed in parallel: Microarray gene-expression profiling (GEP) according to Roberts et al, NEJM 2014; fluorescence in situ hybridization (FISH) with customized probes for ABL2, TSLP, CSF1R, PDGFRB, PTK2B, JAK2, ETV6, IGH, NTRK3, EPOR, TYK2, and IL2RB; and RT-PCR for detection of ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2; quantitative real-time PCR for evaluation of CRLF2-expression, fragment analysis for detection of IKZF1 deletions; next generation sequencing for mutations in JAK1, JAK2, JAK3, KRAS, and NRAS.Results: Based on GEP, we identified 13/132 adult precursor B-ALL pts (10%) as Ph-like ALL. As an alternative approach, we applied a combination of FISH, RT-PCR, fragment analysis and next generation sequencing for diagnosis of Ph-like ALL and simultaneous identification of therapy sensitive targets. Quantitative real time PCR identified 11/132 cases with CRLF2 overexpression (8%), of which 9 cases were also classified as Ph-like ALL according to GEP. 7/11 (64%) cases with elevated CRLF2 expression harbored IGH-CRLF2 rearrangement detected by our FISH assay. Additionally, RT-PCR and FISH analysis identified 4 pts with rearrangements involving TKI sensitive genes ABL1 (SNX2-ABL1; n=1), and PDGFRB (EBF1-PDGFRB; n=3). Rearrangements of EPOR and JAK2 were not detected. Taken together, the combination of quantitative RT-PCR, FISH and fusion specific RT-PCR identified 15 cases (12%) compared to 13 cases (10%) identified by GEP with Ph-like ALL in a cohort of 132 preselected adult precursor B-ALL cases: 13 pts were assigned to the Ph-like subset by both approaches, while no patient was identified by GEP only and two were identified by the alternative approach only.Mutations in JAK2 were detected in 8/15 Ph-like ALL cases, all of which were characterized by CRLF2 rearrangements and/or overexpression of CRLF2. In comparison, none of the 117/132 non Ph-like ALL cases harbored JAK2 mutations. NRAS mutations were found in two pts with CRLF2 rearrangement. Mutations in JAK1, JAK3 and KRAS were not detected. Furthermore, we found a high frequency of IKZF1 deletions in Ph-like ALL pts (n=12/16; 75%) compared to 19% (22/117) in the remaining 117 cases with non Ph-like ALL (p<0.001), which is in line with previous published data.Regarding age, 5/15 Ph-like pts were young adults (age: 31-39 years), 3/15 pts adults (age: 40-55 years) and 7/15 pts were older adults (age: 56-76 years), indicating an incidence of Ph-like ALL of 8% in adults age ≥40 years.Conclusions: In adult precursor B-ALL lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 Ph-like ALL was identified in 10% of pts based on GEP and in 12% by an alternative approach based on RT-PCR and FISH, respectively. As GEP per se is challenging in routine diagnostics and does not provide therapeutic information regarding potential susceptibility to ABL or JAK inhibitors, we postulate the following diagnostic algorithm to identify Ph-like ALL: 1.Analysis of CRLF2 expression.2.FISH targeting ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2.3.Fusion specific RT-PCR for the identification of the respective ABL and JAK fusion partner and sensitive MRD monitoring.We show that the alternative approach is efficient for identification of Ph-like ALL and is feasible for routine diagnostics. DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schlenther:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 1415: Telomere shortening in centroacinar-acinar region of the pancreas: relationships with aging, cancers and tissue stem cells

Abstract Background: Telomeres play a key role in preventing chromosomal instability. There is a striking link between advanced age and increased incidence of pancreatic cancer, suggesting a combined effect of mutation load, epigenetic regulation, telomere dysfunction, and an altered stromal milieu. Pancreatic intraepithelial neoplasia (PanIN) is a major precursor lesion of human pancreatic cancers. However, in genetically engineered mice, there is evidence that pancreatic cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-to-ductal metaplasia (ADM). Determining whether ADM is a precursor lesion of human pancreatic cancers is not clear yet. In this study, we analyzed telomere length in the centroacinar-acinar region of the human pancreas with and without cancer. Methods: We used surgically resected and autopsied pancreatic specimens without cancer (n = 27) and with invasive ductal carcinoma (n = 11). We selected areas without inflammation. Immunohistochemical staining was performed for acinar, ductal, and stem cell markers. Using our quantitative fluorescence in situ hybridization (Q-FISH) technique (Takubo, et al. J Pathol, 2010), we estimated the telomere lengths of acinar and centroacinar cells. Results: Immunohistochemical staining revealed centroacinar cells with cytokeratin (CK) 19 (+)/CK7 (+)/trypsin (-)/CD133 (focal +) and acinar cells with CK19 (-)/CK7 (-)/ trypsin (+)/ CD133 (-). Telomeres in the centroacinar cells was significantly longer than in the acinar cells. In addition, telomere length in centroacinar and acinar cells decreased with age. The rate of decline in telomere length due to age was greater in centroacinar cells than in acinar cells. However, in elderly people at least 60 years of age, telomere length in the centroacinar cells did not decrease, while those in acinar cells continued to decrease after 60 years. The length of telomeres in the centroacinar and acinar cells of pancreatic cancer cases (minimum age, 60 years) was shorter than that in the cells of the cases without cancers. Discussion: Centroacinar cells expressed the stem cell marker, CD133, and had longer telomeres, suggesting that the centroacinar compartments are frequently included among cell types proposed as candidate pancreatic stem. The fact that the maximum regression of telomeres was found in centroacinar cells suggests that the number of stem cells and cells with the longest telomeres decreases with age, as previously described (Aida, et al. Exp Gerontol, 2008). Furthermore, in pancreatic cancer cases, telomere shortening in centroacinar and acinar cells occurs in the early stages of pancreatic carcinogenesis without histological changes. In conclusion, critical telomere dysfunction in the centroacinar-acinar region is possibly due to high annual telomere attrition, which leads to chromosomal instability and carcinogenesis in the pancreas. Citation Format: Yoko Matsuda, Naotaka Izumiyama-Shimomura, Toshiyuki Ishiwata, Mutsunori Fujiwara, Ken-ichiro Tomita, Naoki Hiraishi, Hideki Hamayasu, Ken-ichi Nakamura, Naoshi Ishikawa, Steven Poon, Junko Aida, Kaiyo Takubo, Tomio Arai. Telomere shortening in centroacinar-acinar region of the pancreas: relationships with aging, cancers and tissue stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2015-1415

In situidentification and quantification of starch-hydrolyzing bacteria attached to barley and corn grain in the rumen of cows fed barley-based diets

Cereal grains rich in starch are widely used to meet the energy demands of high-producing beef and dairy cattle. Bacteria are important players in starch digestion in the rumen, and thus play an important role in the hydrolysis and fermentation of cereal grains. However, our understanding of the composition of the rumen starch-hydrolyzing bacteria (SHB) is limited. In this study, BODIPY FL DQ starch staining combined with fluorescence in situ hybridization (FISH) and quantitative FISH were applied to label, identify and quantify SHB possessing active cell-surface-associated (CSA) α-amylase activity in the rumen of heifers fed barley-based diets. When individual cells of SHB with active CSA α-amylase activity were enumerated, they constituted 19-23% of the total bacterial cells attached to particles of four different cultivars of barley grain and corn. Quantitative FISH revealed that up to 70-80% of these SHB were members of Ruminococcaceae in the phylum Firmicutes but were not Streptococcus bovis, Ruminobacter amylophilus, Succinomonas amylolytica, Bifidobacterium spp. or Butyrivibrio fibrisolvens, all of whose amylolytic activities have been demonstrated previously in vitro. The proportion of barley grain in the diet had a large impact on the percentage abundance of total SHB and Ruminococcaceae SHB in these animals.

Telomere shortening in breast cancer correlates with the pathological features of tumor progression.

Telomeres are involved in the maintenance of genomic stability. Telomere alteration has been observed in most human cancer types, and is known to be a feature of malignancy. The aim of the present study was to evaluate whether the telomere length of breast cancer cells correlates with TNM stage and several pathological features. We investigated a total of 44 breast cancers, including 17 scirrhous, 15 papillotubular and 12 solid-tubular carcinomas. Telomere lengths were determined by tissue quantitative fluorescence in situ hybridization (Q-FISH), and compared according to the TNM stage, histological tumor size, lymph node metastases, vascular invasion and immunohistochemical status (ER, PR, HER2 status and Ki67 labeling index). In all histological types, telomeres of cancer cells were significantly shorter than those of normal epithelial cells. Mean telomere length was significantly less in patients with TNM stage III, and in those with large tumors, lymph node metastases and vascular invasion. Our results suggest that the telomere length of cancer cells is strongly correlated with the degree of cancer progression.

Evaluation of Quantitative Fluorescence in situ Hybridization for Relative Measurement of Telomere Length in Placental Mesenchymal Core Cells

Background: Reduced telomere length in placental mesenchymal core cells has been reported during pregnancies complicated by intrauterine growth restriction. To estimate telomere length, a precise, accurate and reproducible technique must be used. Objective: We evaluated the characteristics of a quantitative fluorescence in situ hybridization (Q-FISH) technique for measuring relative telomere length in placental mesenchymal core cells. Methods: From late chorionic villus samplings, telomere length in placental mesenchymal core cells was estimated by a Q-FISH technique using peptide nucleic acid telomere probes. The main characteristics of the Q-FISH technique, such as precision and reproducibility, were evaluated. Results: The telomere length of the cultured placental mesenchymal cells did not follow a normal distribution. When the Q-FISH technique was performed on interphase nuclei of uncultured mesenchymal core cells, normal telomere length distribution was observed. The precision of the technique when applied to cultured placental mesenchymal core cells was estimated to be <6%, and its reproducibility ranged from to 92.9 to 104.7%. Conclusion: Our results showed that cell culture of placental villi produced a non-normal telomere length distribution, probably related to telomere DNA replication during the cell cycle. Despite the influence of cell culture, the Q-FISH technique reported herein showed good precision and reproducibility.

Analysis of EML4-ALK gene fusion mutation in patients with non-small cell lung cancer

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是肺癌的主要类型，相关位点突变检测研究已经成为肺癌分子靶向治疗的热门方向，研究NSCLC肿瘤组织中动物微管相关蛋白4与间变性淋巴瘤激酶融合基因（echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase, EML4-ALK）与表皮生长因子受体（epidermal growth factor receptor, EGFR）的基因突变状态，比较免疫组织化学（immunohistochemistry, IHC）与蝎形探针扩增阻滞突变系统（Scorpions amplification refractory mutation system, Scorpions ARMS）荧光定量PCR与荧光原位杂交（fluorescence in situ hybridization, FISH）检测EML4-ALK融合基因与EGFR基因突变的敏感性。方法应用IHC、ARMS荧光定量PCR及FISH技术检测85例NSCLC石蜡包埋肿瘤组织以及癌旁正常肺组织中EML4-ALK融合基因状态，并应用ARMS方法检测EGFR基因第18、19、20和21外显子突变状态。结果115例NSCLC中IHC显示32例有ALK（D5F3）表达，表达率为27.8%;ARMS检测27例存在EML4-ALK融合基因突变，突变检出率为23.5%;53例检出EGFR突变，突变率为46%。而FISH检测23例存在EML4-ALK融合基因突变，检出率为20%，稍低于ARMS检测结果，提示ARMS的敏感度更高。结论联合运用IHC/ARMS荧光定量PCR/FISH技术能够对EML4-ALK融合基因状态做出快速、准确评价。

Optimization of Retention Time of Microbial Community Structure of Activated Sludge Process

Ammonia Oxidizing Bacteria community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT did not reduce the nitrification activity, but the number per mixed liquor suspended solids nor was community composition of AOB affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition.

Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full-scale wastewater treatment plants.

Numerous past studies have shown members of the genus Nitrospira to be the predominant nitrite-oxidizing bacteria (NOB) in nitrifying wastewater treatment plants (WWTPs). Only recently, the novel NOB 'Candidatus Nitrotoga arctica' was identified in permafrost soil and a close relative was enriched from activated sludge. Still, little is known about diversity, distribution and functional importance of Nitrotoga in natural and engineered ecosystems. Here we developed Nitrotoga 16S rRNA-specific PCR primers and fluorescence in situ hybridization (FISH) probes, which were applied to screen activated sludge samples from 20 full-scale WWTPs. Nitrotoga-like bacteria were detected by PCR in 11 samples and reached abundances detectable by FISH in seven sludges. They coexisted with Nitrospira in most of these WWTPs, but constituted the only detectable NOB in two systems. Quantitative FISH revealed that Nitrotoga accounted for nearly 2% of the total bacterial community in one of these plants, a number comparable to Nitrospira abundances in other WWTPs. Spatial statistics revealed that Nitrotoga coaggregated with ammonia-oxidizing bacteria, strongly supporting a functional role in nitrite oxidation. This activity was confirmed by FISH in combination with microradiography, which revealed nitrite-dependent autotrophic carbon fixation by Nitrotoga in situ. Correlation of the presence or absence with WWTP operational parameters indicated low temperatures as a main factor supporting high Nitrotoga abundances, although in incubation experiments these NOB remained active over an unexpected range of temperatures, and also at different ambient nitrite concentrations. In conclusion, this study demonstrates that Nitrotoga can be functionally important nitrite oxidizers in WWTPs and can even represent the only known NOB in engineered systems.

P05.02 * KIAA1549:BRAF FUSION GENE IN PEDIATRIC BRAIN TUMORS OF VARIOUS HISTOGENESIS

INTRODUCTION: Pediatric GBMs (pGBM) are histologically indistinguishable from adult GBM (aGBM) however they are molecularly distinct. Few molecular data exists on the mechanism underlying development and progression of pGBM, whereas a number of gene expression profiling analyses performed in aGBM helped to identify molecular events leading to oncogenesis and provided more accurate means for classification of subtypes, outcomes and even response to treatments. The KIAA1549:BRAF fusion (K-B) gene has been considered a driver of genetic events and a useful diagnostic marker in adult pilocytic astrocytoma (PA). The K-B fusion, on the other hand, is an unusual finding in pediatric diffuse astrocytomas and oligoastocytoma and not reported in pHGG On the other hand, it has been reported a low frequency of B_F fusion gene in adult diffuse gliomas in association with IDH1 mutations. METHODS: To further elucidate the possible occurrence of the K-B fusion gene in non-PA tumors, we investigated its presence in a series of 69 pediatric brain neoplasms of different histogenesis and grade (34 non PA and 35 PA). Fusion has been investigated through the analysis of messenger RNA by reverse-transcription polymerase chain reaction (RT-PCR) followed by sequencing. Selected cases were screened for mutational analysis for IDH1, BRAFv600 and H3F3A. Positive cases have undergone to confirmation by the interphase Fluorescence in situ Hybridization (FISH) analysis and quantitative PCR (Taqman). RESULTS: We detected the KIAA1549:BRAF fusion (K-B) gene in 5 out of 34 (14.7%) non-PA primary tumors, i.e. 1 glioblastoma, 1 anaplastic astrocytoma, 1 anaplastic PXA, 1 ependymoma and 1 AT/RT. The fusion gene was also found in 21 out of 35 PAs (63.6%) and in 1/3 (33%) WHO grade I gangliogliomas. Quantitative PCR and FISH analysis confirmed the presence of the K-B fusion gene. The frequency of fusion harboring nuclei in non-PA neoplasms was lower than in PAs. Screening for IDH1, BRAFv600, and H3F3A mutations disclosed one case with the IDH1 and one with the K27M-H3.3 mutation, both unassociated with the K-B gene. CONCLUSION: Our study indicates that: 1) the K-B fusion gene can occasionally be detected in non-PA tumors of various histogenesis; 2) its occurrence in these neoplasms is probably due rather to a random alteration related to genomic instability than to a driving molecular event, as in PAs; and 3) the K-B fusion gene's role as a diagnostic marker outside the histological spectrum of pediatric low-grade gliomas should be carefully considered.

Arm-specific telomere dynamics of each individual chromosome in induced pluripotent stem cells revealed by quantitative fluorescence in situ hybridization.

We have reported that telomere fluorescence units (TFUs) of established induced pluripotent stem cells (iPSCs) derived from human amnion (hAM933) and fetal lung fibroblasts (MRC-5) were significantly longer than those of the parental cells, and that the telomere extension rates varied quite significantly among clones without chromosomal instability, although the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased along with chromosomal abnormalities from an early stage. In the present study we attempted to clarify telomere dynamics in each individual chromosomal arm of parental cells and their derived clonal human iPSCs at different numbers of passages using quantitative fluorescence in situ hybridization (Q-FISH). Although no specific arm of any particular chromosome appeared to be consistently shorter or longer than most of the other chromosomes in any of the cell strains, telomere elongation in each chromosome of an iPSC appeared to be random and stochastic. However, in terms of the whole genome of any specific cell, the telomeres showed overall elongation associated with iPSC generation. We have thus demonstrated the specific telomere dynamics of each individual chromosomal arm in iPSCs derived from parental cells, and in the parental cells themselves, using Q-FISH.

Different TP53 mutations are associated with specific chromosomal rearrangements, telomere length changes, and remodeling of the nuclear architecture of telomeres.

TP53 mutations are the most common mutations in human cancers, and TP53-R175H and TP53-R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53-V143A, TP53-R175H, and TP53-R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three-dimensional (3D) quantitative FISH (Q-FISH) on interphase and Q-FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53-R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53-R175H and TP53-V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53-R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53-R175H. Taken together, our results indicate that the mutation TP53-R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major determinants of the TP53-dependent genomic instability.

Q-FISH measurement of hepatocyte telomere lengths in donor liver and graft after pediatric living-donor liver transplantation: donor age affects telomere length sustainability.

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Furthermore, since contradictory data have been reported with regard to the relationship between donor age and LT/LDLT outcome, the question of whether the use of elderly donors influences the long-term outcome of a graft after LT/LDLT remains unsettled. To address whether hepatocyte telomere length reflects the outcome of LDLT, we analyzed the telomere lengths of hepatocytes in informative biopsy samples from 12 paired donors and recipients (grafts) of pediatric LDLT more than 5 years after adult-to-child LDLT because of primary biliary atresia, using quantitative fluorescence in situ hybridization (Q-FISH). The telomere lengths in the paired samples showed a robust relationship between the donor and grafted hepatocytes (r = 0.765, p = 0.0038), demonstrating the feasibility of our Q-FISH method for cell-specific evaluation. While 8 pairs showed no significant difference between the telomere lengths for the donor and the recipient, the other 4 pairs showed significantly shorter telomeres in the recipient than in the donor. Multiple regression analysis revealed that the donors in the latter group were older than those in the former (p = 0.001). Despite the small number of subjects, this pilot study indicates that donor age is a crucial factor affecting telomere length sustainability in hepatocytes after pediatric LDLT, and that the telomeres in grafted livers may be elongated somewhat longer when the grafts are immunologically well controlled.

Telomere shortening and increased oxidative stress are restricted to venous tissue in patients with varicose veins: A merely local disease?

Shortened telomere length (TL) and oxidative stress have been described in several vascular disorders at both the tissue and circulating level. However, to our knowledge, there are no reports about TL associated with varicose vein (VV) disease. This paper aimed to evaluate, at the tissue and circulating level, TL and oxidative stress in VV disease, compared to the corresponding counterparts from abdominal aortic aneurysm (AAA) patients and control healthy subjects. TL was measured using quantitative fluorescence in situ hybridization (Q-FISH). Oxidative stress was evaluated by measuring the malondialdehyde (MDA) concentration by thiobarbituric acid reactive substance/s (TBARS) assay. At the vascular tissue level, VV patients had shortened TL and a high MDA concentration, similarly to AAA patients. Conversely, blood lymphocytes and epidermal cells from VV patients had a TL similar to healthy controls and significantly longer than the same cells from AAA patients. Moreover, the MDA concentration in plasma from VV patients was significantly lower than from the AAA group. Linear regression analysis showed a statistically significant inverse correlation between the blood lymphocyte TL and plasma MDA level. Our results suggest that, unlike AAA, telomere attrition in VV tissue is not a systemic phenomenon but it may be attributable to tissue microenvironment conditions and possibly to high local oxidative stress.

Telomere shortening and telomere position effect in mild ring 17 syndrome

BackgroundRing chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations.ResultsThe results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family.ConclusionsSubtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

Key Role of Promoter Methylation and Inactivation of MCPH1 Gene in Brain Tumors

Background: Microcephalin (MCPH1/BIRT1) is a putative tumor suppressor gene which has recently seized great attentions in cancer studies. The present study was conducted to seek the impact of MCPH1 promoter methylation on development of brain tumors and telomere repeat length (TRL). Methods: The brain tissue section provided from brain tumor patients and two normal brain autopsies were undergone DNA isolation. DNA samples treated with bisulfite sodium using DNA modification kit (Qiagen) were amplified in methylation specific polymerization chain reaction (MSP-PCR) confirmed by sequencing. Protein expression analysis was performed by immunofluorescence (IF) assay using antibodies against MCPH1, cyclin E and CDC25A proteins. The TRL of brain tumor patients was determined through quantitative fluorescent in situ hybridization (Q-FISH). Results: The MCPH1 gene promoter was methylated in 96.6% of the patients consistent with protein expression pattern and the telomere statue was confirmed by low or absence of signal in tumor cells. Conclusion: MCPH1 promoter methylation had strong association with TRL, tumor’s grade and stage (P < 0.05). TRL was meaningfully associated with grade and subtype pathology of brain tumors (P = 0.01). Further studies are required to clarify the exact role of MCPH1 gene on TRL and tumor suppression especially in brain tumors. J Neurol Res. 2014;4(5-6):133-138 doi: http://dx.doi.org/10.14740/jnr295e

Key Role of Promoter Methylation and Inactivation of MCPH1 Gene in Brain Tumors

Background: Microcephalin (MCPH1/BIRT1) is a putative tumor suppressor gene which has recently seized great attentions in cancer studies. The present study was conducted to seek the impact of MCPH1 promoter methylation on development of brain tumors and telomere repeat length (TRL). Methods: The brain tissue section provided from brain tumor patients and two normal brain autopsies were undergone DNA isolation. DNA samples treated with bisulfite sodium using DNA modification kit (Qiagen) were amplified in methylation specific polymerization chain reaction (MSP-PCR) confirmed by sequencing. Protein expression analysis was performed by immunofluorescence (IF) assay using antibodies against MCPH1, cyclin E and CDC25A proteins. The TRL of brain tumor patients was determined through quantitative fluorescent in situ hybridization (Q-FISH). Results: The MCPH1 gene promoter was methylated in 96.6% of the patients consistent with protein expression pattern and the telomere statue was confirmed by low or absence of signal in tumor cells. Conclusion: MCPH1 promoter methylation had strong association with TRL, tumor’s grade and stage (P &lt; 0.05). TRL was meaningfully associated with grade and subtype pathology of brain tumors (P = 0.01). Further studies are required to clarify the exact role of MCPH1 gene on TRL and tumor suppression especially in brain tumors. J Neurol Res. 2014;4(5-6):133-138 doi: http://dx.doi.org/10.14740/jnr295e

A new der(1;7)(q10;p10) leading to a singular 1p loss in a case of glioblastoma with oligodendroglioma component

The combined 1p-/19q- deletions in oligodendrogliomas originate from translocation between both chromosomes. In the few cases of oligoastrocytomas and glioblastomas with an oligodendroglioma component (GBMO) where only 1p deletion was described, the origin remains unknown. We report the first case of GBMO, in which a single 1p deletion was detected and was linked to a translocation between chromosomes 1 and 7. Fresh surgical specimens were collected during surgery and the samples were used for cell culture, touch preparation smear slides (TP slides) and DNA extraction. Peripheral venous blood was also collected from the patient. G-banding using Trypsin and stained with Giemsa (GTG) banding and karyotyping were performed and 1p-/19q-, TP53, PTEN and c-MYC were analyzed by fluorescent in situ hybridization (FISH). Multicolor FISH (mFISH) and microsatellites analyses were also performed to complete the investigation. Three-dimensional quantitative FISH (3D-QFISH) of telomeres was performed on nuclei from TP slides and analyzed using TeloView(TM) to determine whether the 3D telomere profile as an assessment of telomere dysfunction and a characterization of genomic instability could predict the disease aggressiveness. An unbalanced chromosomal translocation was found in all metaphases and confirmed by mFISH. The karyotype of the case is: 50∼99,XXX, +der(1;7)(q10;p10),inc[47] The derivative chromosome was found in all 47 analyzed cells, but the number of derivatives varied from one to four. There was neither imbalance in copy number for genes TP53 and PTEN, nor amplification of c-MYC gene. We did not find loss of heterozygosity with analysis of microsatellite markers for chromosomes 1p and 19q in tumor cells. The 3D-telomere profile predicted a very poor prognostic and short-term survival of the patient and highlights the potential clinical power of telomere signatures as a solid biomarker of GBMO. Furthermore, this translocation between chromosomes 1 and 7 led to a singular 1p deletion in this GBMO and may generate the 1p and 7q deletions.