Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In this study, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, proC and rpoD formed the most stable pair (r = 0.958; P < 0.001). Next, in four ciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA was significantly up-regulated, while the oprD gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore lends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.

Imatinib mesylate (IM), 400 mg daily, is the standard treatment of Philadelphia-positive (Ph⁺) chronic myeloid leukemia (CML). Preclinical data and results of single-arm studies raised the suggestion that better results could be achieved with a higher dose. To investigate whether the systematic use of a higher dose of IM could lead to better results, 216 patients with Ph⁺ CML at high risk (HR) according to the Sokal index were randomly assigned to receive IM 800 mg or 400 mg daily, as front-line therapy, for at least 1 year. The CCgR rate at 1 year was 64% and 58% for the high-dose arm and for the standard-dose arm, respectively (P = .435). No differences were detectable in the CgR at 3 and 6 months, in the molecular response rate at any time, as well as in the rate of other events. Twenty-four (94%) of 25 patients who could tolerate the full 800-mg dose achieved a CCgR, and only 4 (23%) of 17 patients who could tolerate less than 350 mg achieved a CCgR. This study does not support the extensive use of high-dose IM (800 mg daily) front-line in all CML HR patients. This trial was registered at www.clinicaltrials.gov as #NCT00514488.

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20–30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, P_meta = 2.5 × 10⁻⁹, OR[T] = 0.81) and 17q21.32 (rs72823592, P_meta = 9.3 × 10⁻⁹, OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, P_meta = 9.1 × 10⁻⁹, OR[T] = 0.68) and at 1q43 for JME (rs12059546, P_meta = 4.1 × 10⁻⁸, OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, P_meta = 4.0 × 10⁻⁶) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndromes.

Multiple genes have been shown to be independently hypermethylated in lymphoid malignancies. We report here on the extent of concurrent methylation of E-cadherin, Dap-kinase, O⁶MGMT, p73, p16, p15 and p14 in 129 pediatric ALL cases. While most of these genes demonstrated methylation in a proportion of cases, O⁶MGMT, p16 and p14 were infrequently methylated (11, 7 and 3%, respectively). Methylation of at least one gene was found in the vast majority (83%) of cases. To determine the extent and concordance of methylation we calculated a methylation index (MI=number of methylated genes/number of studied genes) for each sample. The average MI was 0.28, corresponding to 2/7 methylated genes. MI was correlated with standard prognostic factors, including immunophenotype, age, sex, WBC and presence of specific translocations (TEL-AML1, BCR-ABL, E2A-PBX1 or MLL-AF4). We determined that children ⩾ 10 years old and children presenting with high WBC ( ⩾ 50 × 10⁹/l) both associated with a higher MI (P<0.01 and <0.05, respectively). T-ALLs demonstrated a lower MI (median=0.17) than precursor B ALLs (median=0.28). Among the different molecular subgroups, MLL-ALLs had the highest MI (mean=0.35), while ALLs carrying the t(1;19) had the lowest MI (mean=0.07). The most common epigenetic lesion in childhood ALL was methylation of E-cadherin (72%) independent of the molecular subtype or other clinicopathological factors.