Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs.

Clinical score of 62 Italian patients with Cornelia de Lange syndrome and correlations with the presence and type of NIPBL mutation.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, upper limb abnormalities, growth and cognitive retardation. About half of all patients with CdLS carry mutations in the NIPBL gene. The first Italian CdLS cohort involving 62 patients (including 4 related members) was screened for NIPBL mutations after a clinical evaluation using a quantitative score that integrates auxological, malformation and neurodevelopmental parameters. The patients were classified as having an overall 'severe', 'moderate' or 'mild' phenotype. NIPBL screening showed 26 mutations so classified: truncating (13), splice-site (8), missense (3), in-frame deletion (1) and regulatory (1). The truncating mutations were most frequently found in the patients with a high clinical score, whereas most of the splice-site and all missense mutations clustered in the low-medium score groups. The NIPBL-negative group included patients covering the entire clinical spectrum. The prevalence of a severe phenotype in the mutated group and a mild phenotype in the non-mutated group was statistically significant. In terms of the isolated clinical signs, the statistically significant differences between the mutation-positive and mutation-negative individuals were pre- and post-natal growth deficits, limb reduction, and delayed speech development. The proposed score seems to be a valuable means of prioritizing the patients with CdLS to undergo an NIPBL mutation test.

Evidence by expression analysis of candidate genes for congenital heart defects in the NF1 microdeletion interval.

It was recently reported that congenital heart disease is significantly more frequent in patients with NF1 microdeletion syndrome than in those with classical NF1. The outcome of congenital heart disease in this subset of patients is likely caused by the haploinsufficiency of gene/s in the deletion interval. Following in silico analysis of the deleted region, we found two genes known to be expressed in adult heart, the Joined to JAZF1 (SUZ12) and the Centaurin-alpha 2 (CENTA2) genes, and seven other genes with poorly defined patterns of expression and function. With the aim of defining their expression profiles in human fetal tissues (15th-21st weeks of gestation), expression analysis by RT-PCR and Northern blotting was performed. C17orf40, SUZ12 and CENTA2 were found to be mainly expressed in fetal heart, and following RT-PCR on mouse embryos and embryonic heart and brain at different stages of development, we found that the orthologous genes C17orf40, Suz12 and Centa2 are also expressed in early stages of development, before and during the formation of the four heart chambers. The presence of binding sites for Nkx2-5, a transcription factor expressed early in heart development, in all three mouse orthologous genes was predicted by bioinformatics, thus reinforcing the hypothesis that these genes might be involved in heart development and may be plausible candidates for congenital heart disease.

Identification of duplicated genes in 17q11.2 using FISH on stretched chromosomes and DNA fibers.

Recent evidence has been provided that links duplicons (REP-P and REP-M) in 17q11.2 flanking the neurofibromatosis type 1 (NF1) gene to the breakpoints of the NF1 microdeletion syndrome. The physical mapping and structural definition of duplicated regions is often impossible by conventional approaches, and so we have used high resolution fluorescence in situ hybridization (FISH) with locus-specific probes of limited size on chromosomes stretched to different degrees to identify novel duplicated genes and expressed sequence tags (ESTs) mapping to 17q11.2. This approach has allowed us to detect and map duplications of the BLMH and GOS28 genes, with one copy lying centromeric and one telomeric to the NF1 gene, and an SHGC30113 transcript with one copy being adjacent and the other distal to the NF1 3' untranslated region. Double-color FISH with a BLMH-specific probe on stretched chromosomes showed that the telomeric BLMH repeat lacked the 5' end of the gene and was 0.8 Mb from its centromeric copy. The distance between the SHGC30113 repeats was estimated as being 500 kb by double-color FISH on highly stretched chromosomes and DNA fibers. The latter approach revealed adjacent SHGC30113-BLMH-specific signals relating to the incomplete BLMH copy. The use of FISH on stretched chromosomes and, where applicable DNA fibers, is a powerful tool for identifying and finely characterizing duplicated regions whose mapping by the classical physical mapping approach is often precluded.