Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

In order to contribute to the knowledge of type and frequency of chromosome abnormalities in early pregnancy losses, we analyzed the cytogenetic results from a large series of first trimester miscarriages, using a diagnostic approach with a high success rate and no maternal contamination. A total of 1,119 consecutive chorionic villi samples were obtained before evacuation, and karyotypes were prepared after short-term culture (STC). In 603 samples, a long-term culture (LTC) was also performed. The overall and individual frequencies of the different types of chromosome abnormalities were established, including placental mosaicisms, and their relationship with maternal age and gestational weeks was assessed. An abnormal karyotype was detected in 70.3% of the samples. Single autosomal trisomy was the most frequent abnormality (64.6% of the abnormal cases), followed by triploidy (13.1%) and monosomy X (10.4%). Chromosome rearrangements were found in 5.2%, combined abnormalities in 8.9%, and placental mosaicism in 3.5% of the cases with STC and LTC performed. Individual trisomies behaved differently with respect to maternal age and intrauterine survival. Due to the combination of STC and LTC, our study offers reliable information on the incidence and type of chromosome abnormalities and placental mosaicism in miscarriages and contributes to define the cytogenetic implication in their etiology.

In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)--a new copper compound exhibiting antineoplastic activity--on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-L-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by macroglossia, macrosomia, and abdominal wall defects. It is a multigenic disorder caused in most patients by alterations in growth regulatory genes. A small number of individuals with BWS (5-10%) have mutations in CDKN1C, a cyclin-dependent kinase inhibitor of G1 cyclin complexes that functions as a negative regulator of cellular growth and proliferation. Here, we report on eight patients with BWS and CDKN1C mutations and review previous reported cases. We analyzed 72 patients (50 BWS, 17 with isolated hemihyperplasia (IH), three with omphalocele, and two with macroglossia) for CDKN1C defects with the aim to search for new mutations and to define genotype-phenotype correlations. Our findings suggest that BWS patients with CDKN1C mutations have a different pattern of clinical malformations than those with other molecular defects. Polydactyly, genital abnormalities, extra nipple, and cleft palate are more frequently observed in BWS with mutations in CDKN1C. The clinical observation of these malformations may help to decide which genetic characterization should be undertaken (i.e., CDKN1C screening), thus optimizing the laboratory evaluation for BWS.

No competing interests declared.

Background: Aproximately 5-10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects.Results: Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion:This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients.

Objectives

Nuclear transfer experiments undertaken in the mid-80's revealed that both maternal and paternal genomes are necessary for normal development. This is due to genomic imprinting, an epigenetic mechanism that results in parent-of-origin monoallelic expression of genes regulated by germline-derived allelic methylation. To date, ∼100 imprinted transcripts have been identified in mouse, with approximately two-thirds showing conservation in humans. It is currently unknown how many imprinted genes are present in humans, and to what extent these transcripts exhibit human-specific imprinted expression. This is mainly due to the fact that the majority of screens for imprinted genes have been undertaken in mouse, with subsequent analysis of the human orthologues. Utilizing extremely rare reciprocal genome-wide uniparental disomy samples presenting with Beckwith-Wiedemann and Silver-Russell syndrome-like phenotypes, we analyzed ∼0.1% of CpG dinculeotides present in the human genome for imprinted differentially methylated regions (DMRs) using the Illumina Infinium methylation27 BeadChip microarray. This approach identified 15 imprinted DMRs associated with characterized imprinted domains, and confirmed the maternal methylation of the RB1 DMR. In addition, we discovered two novel DMRs, first, one maternally methylated region overlapping the FAM50B promoter CpG island, which results in paternal expression of this retrotransposon. Secondly, we found a paternally methylated, bidirectional repressor located between maternally expressed ZNF597 and NAT15 genes. These three genes are biallelically expressed in mice due to lack of differential methylation, suggesting that these genes have become imprinted after the divergence of mouse and humans.