NGS and blood group systems: State of the art and perspectives.

Molecular analysis, or genotyping, of genes involved in the expression of blood group antigens has been a standard strategy used in immunohaematology laboratories routinely. For the past ten years, next-generation sequencing (NGS), or second-generation sequencing, has become the reference method in genetics. Extensive study of distinct targets, large genomic regions, and even whole genome is henceforth possible by this approach at minimal cost. Blood group genotyping has thus taken advantage of this technological advent. A few preliminary studies have open the way to NGS in this field by studying one or several genes, in a wide range of samples (donors and patients) by using several different platforms. These works have helped in the identification of both the benefits and limitations of the technology. Other recently published studies have benefited from these preliminary data to improve the methodology, specificity and accuracy of output data. In parallel novel strategies, i.e. third-generation sequencing, which can sequence long DNA regions at the single-molecule level, have emerged and shown promise for the potential resolution of complex rearrangements involving genes of the Rh and MNS blood group systems respectively. As technological and methodological hurdles have been overcome, these approaches may be used in a clinical situation in a near future.

Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes.

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene that encodes the methyl-CpG-binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild-type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome-wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild-type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non-sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT-PCR indicated four up-regulated genes and five down-regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up-regulated candidate genes and sequencing of bisulphite-converted DNA indicated that MeCP2 preferentially binds to methylated-DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.

KLN-5: a safe monocationic lipophosphoramide to transfect efficiently haematopoietic cell lines and human CD34+ cells.

The safe and efficient delivery of nucleic acids into haematopoietic stem cells (HSCs) has a wide range of therapeutic applications. Although viruses are being used in most clinical trials owing to their high transfection efficacy, recent results highlight many concerns about their use. Synthetic transfection reagents, in contrast, have the advantage of being safe and easy to manage while their low transfection efficiency remains a hurdle that needs to be addressed before they can be widely used. Using information on transfection mechanisms, a new family of monocationic lipids called lipophosphoramides was synthesized. Their efficiency to transfer genes into haematopoietic cell lines (K562, Jurkat and Daudi) and CD34+ cells was assessed. In this study, we report that one of these new compounds, KLN-5, leads to more efficient transfection activity than one of our previously most efficient reagents (EG-308) and the commercially available monocationic lipids (DC-CHOL and DOTAP/DOPE) (P<0.05). In addition, only a slight toxicity related to the chemical structure of the new compounds is observed. Moreover, we show that KLN-5 can successfully carry the transgene into haematopoietic progenitor cells (CD34+). These results demonstrate that synthetic transfection reagents represent a viable alternative to viruses and could have potential practical utility in a number of applications.