Efficient QTL detection for nonhost resistance in wild lettuce: backcross inbred lines versus F2 population

In plants, several population types [F2, recombinant inbred lines, backcross inbred lines (BILs), etc.] are used for quantitative trait locus (QTL) analyses. However, dissection of the trait of interest and subsequent confirmation by introgression of QTLs for breeding purposes has not been as successful as that predicted from theoretical calculations. More practical knowledge of different QTL mapping approaches is needed. In this recent study, we describe the detection and mapping of quantitative resistances to downy mildew in a set of 29 BILs of cultivated lettuce (L. sativa) containing genome segments introgressed from wild lettuce (L. saligna). Introgression regions that are associated with quantitative resistance are considered to harbor a QTL. Furthermore, we compare this with results from an already existing F2 population derived from the same parents. We identified six QTLs in our BIL approach compared to only three in the F2 approach, while there were two QTLs in common. We performed a simulation study based on our actual data to help us interpret them. This revealed that two newly detected QTLs in the BILs had gone unnoticed in the F2, due to a combination of recessiveness of the trait and skewed segregation, causing a deficit of the wild species alleles. This study clearly illustrates the added value of extended genetic studies on two different population types (BILs and F2) to dissect complex genetic traits

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell line

MGMT promoter hypermethylation is a frequent, early, and consistent event in astrocytoma progression, and not correlated with TP53 mutation

Hypermethylation of the MGMT gene promoter and mutation of the TP53 tumor-suppressor gene are frequently present in diffuse astrocytomas. However, there is only anecdotal information about MGMT methylation status and TP53 mutations during progression of low-grade diffuse astrocytoma (AII) to anaplastic astrocytoma (AIII) and secondary glioblastoma (sGB). In this study biopsy specimens from 51 patients with astrocytic tumors with radiologically proved progression from low to high-grade malignancy were investigated for the presence and consistency of MGMT promoter hypermethylation and TP53 mutations. For 27 patients biopsy samples both of primary tumors and their recurrences were available. For the other 24 patients histology of either the low-grade lesion or the high-grade recurrence was available. It was found that MGMT promoter hypermethylation and TP53 mutations are both frequent and early events in the progression of astrocytomas and that their status is consistent over time. No correlation was found between MGMT methylation status and the presence of TP53 mutations. In addition, no correlation was found between MGMT promoter hypermethylation and the type of TP53 mutations. These results argue against the putative TP53 G:C>A:T transition mutations suggested to occur preferentially in MGMT hypermethylated tumors

Hypersensitive Response-Like Reaction Is Associated with Hybrid Necrosis in Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii Coss

BACKGROUND: Hybrid speciation is classified into homoploid and polyploid based on ploidy level. Common wheat is an allohexaploid species that originated from a naturally occurring interploidy cross between tetraploid wheat and diploid wild wheat Aegilops tauschii Coss. Aegilops tauschii provides wide naturally occurring genetic variation. Sometimes its triploid hybrids with tetraploid wheat show the following four types of hybrid growth abnormalities: types II and III hybrid necrosis, hybrid chlorosis, and severe growth abortion. The growth abnormalities in the triploid hybrids could act as postzygotic hybridization barriers to prevent formation of hexaploid wheat. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report on the geographical and phylogenetic distribution of Ae. tauschii accessions inducing the hybrid growth abnormalities and showed that they are widely distributed across growth habitats in Ae. tauschii. Molecular and cytological characterization of the type III necrosis phenotype was performed. The hybrid abnormality causing accessions were widely distributed across growth habitats in Ae. tauschii. Transcriptome analysis showed that a number of defense-related genes such as pathogenesis-related genes were highly up-regulated in the type III necrosis lines. Transmission electron microscope observation revealed that cell death occurred accompanied by generation of reactive oxygen species in leaves undergoing type III necrosis. The reduction of photosynthetic activity occurred prior to the appearance of necrotic symptoms on the leaves exhibiting hybrid necrosis. CONCLUSIONS/SIGNIFICANCE: Taking these results together strongly suggests that an autoimmune response might be triggered by intergenomic incompatibility between the tetraploid wheat and Ae. tauschii genomes in type III necrosis, and that genetically programmed cell death could be regarded as a hypersensitive response-like cell death similar to that observed in Arabidopsis intraspecific and Nicotiana interspecific hybrids. Only Ae. tauschii accessions without such inhibiting factors could be candidates for the D-genome donor for the present hexaploid wheat

Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines

Genetic and metabolic predictors of chemosensitivity in oligodendroglial neoplasms

The −1p/−19q genotype predicts chemosensitivity in oligodendroglial neoplasms, but some with intact 1p/19q also respond and not all with 1p/19q loss derive durable benefit from chemotherapy. We have evaluated the predictive and prognostic significance of pretherapy 201Tl and 18F-FDG SPECT and genotype in 38 primary and 10 recurrent oligodendroglial neoplasms following PCV chemotherapy. 1p/19q loss was seen in 8/15 OII, 6/15 OAII, 7/7 OIII, 3/11 OAIII and was associated with response (Fisher-Exact: P=0.000) and prolonged progression-free (log-rank: P=0.002) and overall survival (OS) (log-rank: P=0.0048). Response was unrelated to metabolism, with tumours with high or low metabolism showing response. Increased 18F-FDG or 201Tl uptake predicted shorter progression-free survival (PFS) in the series (log-rank: 201Tl P=0.0097, 18F-FDG P=0.0170) and in cases with or without the −1p/−19q genotype. Elevated metabolism was associated with shorter OS in cases with intact 1p/19q (log-rank: 18F-FDG P=0.0077; 201Tl P=0.0004) and shorter PFS in responders (log-rank: 18F-FDG P=0.005; 201Tl P=0.0132). 201Tl uptake and 1p/19q loss were independent predictors of survival in multivariate analysis. In this initial study, 201Tl and 18F-FDG uptake did not predict response to PCV, but may be associated with poor survival following therapy irrespective of genotype. This may be clinically useful warranting further study

Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poor survival of renal-cell carcinoma

International audienceBACKGROUND: The von Hippel-Lindau gene (VHL) alteration, a common event in sporadic clear-cell renal-cell carcinoma (CCRCC), leads to highly vascularised tumours. Vascular endothelial growth factor (VEGF) is the major factor involved in angiogenesis, but the prognostic significance of both VHL inactivation and VEGF expression remain controversial. The aims of this study were to analyse the relationship between VHL genetic and epigenetic alterations, VHL expression and VEGF tumour or plasma expression, and to analyse their respective prognostic value in patients with CCRCC. METHODS: A total of 102 patients with CCRCC were prospectively analysed. Alterations in VHL were determined by sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA. Expression of pVHL and VEGF was determined by immunohistochemistry. Plasma VEGF was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: VHL mutation, deletion and promoter methylation were identified in 70, 76 and 14 cases, respectively. Overall, at least one VHL-gene alteration occurred in 91 cases (89.2%). Both VEGF tumour and plasma expression appeared to be decreased in case of VHL alteration. Median progression-free survival and CCRCC-specific survival were significantly reduced in patients with wild-type VHL or altered VHL and high VEGF expression, which, therefore, represent two markers of tumour aggressiveness in CCRCC. CONCLUSION: Stratifying CCRCCs according to VHL and VEGF status may help tailor therapeutic strategy

Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference

<p>Abstract</p> <p>Background</p> <p>Epigenetic silencing of the <it>MGMT </it>gene by promoter methylation is associated with loss of <it>MGMT </it>expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.</p> <p>Methods</p> <p>An analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The <it>SNURF </it>promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.</p> <p>Results</p> <p>Concordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). <it>MGMT </it>promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with <it>MGMT </it>unmethylated and methylated, respectively. Considering <it>MGMT </it>methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring <it>MGMT </it>promoter unmethylated and other patients with any percentage of <it>MGMT </it>methylation (p = 0.003). This difference was retained using other cut off values for <it>MGMT </it>methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of <it>MGMT </it>methylated allele was used as cut-off.</p> <p>Conclusions</p> <p>We report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated <it>MGMT </it>alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of <it>MGMT </it>promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.</p

A study of genomic instability in early preneoplastic colonic lesions

It is difficult to explain the differential rates of progression of premalignant colonic lesions and differences in behaviour of morphologically similar lesions. Heterogeneity for microsatellite instability (MSI) and promoter methylation in driving these phenomena forward may explain this; however, no previous analysis has examined this in detail at the gland level, the smallest unit of colorectal premalignant lesions. We aimed to carry out an analysis of gland level genomic instability for MSI and promoter methylation. MSI occurred significantly more frequently (20%) in colonic glands than has previously been observed in whole colorectal polyps. Significant promoter methylation was seen in MLH1,PMS2,MLH3 and MSH3 as well as significant heterogeneity for both MSI and promoter methylation. Methylation and MSI may have a significant role in driving forward colorectal carcinogenesis, although in the case of MSI, this association is less clear as it occurs significantly more frequently than previously thought, and may simply be a passenger in the adenoma-carcinoma sequence. Promoter methylation in MLH1,MLH3,MSH3 and PMS2 was also found to be significantly associated with MSI and should be investigated further. A total of 273 colorectal glands (126 hyperplastic, 147 adenomatous) were isolated via laser capture microdissection (targeted at regions of MLH1 loss) from 93 colonic polyps and tested for MSI, and promoter methylation of the DNA mismatch repair genes MLH1,MSH2,MLH3,MSH6,PMS2,MGMT and MLH3 via methylation specific multiplex ligation-dependent probe amplification. Logistic regression modelling was then used to identify significant associations between promoter methylation and gland histological type and MSI status