Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4–6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5′) splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3′) splice site. cDNA analysis showed the inclusion of a 212-bp non-coding ‘pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction

Molecular Evidence of Lentiviral Vector-Mediated Gene Transfer into Human Self-Renewing, Multi-potent, Long-Term NOD/SCID Repopulating Hematopoietic Cells

A major challenge in gene therapy is to achieve efficient transduction of hematopoietic stem cells (HSC). It has previously been shown that lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). Here we studied the effect of cytokines during the short ex vivo incubation with vector. Although SRC transduction was efficient without stimulation, the presence of cytokines significantly improved it. The treatment did not affect the engraftment level or the SRC frequency, but seemed to enhance SRC susceptibility to LV. SRC transduced in both conditions repopulated primary and secondary recipients, maintaining stable multi-lineage transgene expression. Using linear amplification-mediated PCR, we then analyzed vector integration in the bone marrow and CFC of the engrafted mice to monitor the clonal activity of the transduced SRC in vivo. We showed polyclonal engraftment, multi-lineage differentiation, and propagation to secondary recipients of individual SRC. We observed multiple integrations in most clones. These results provide the first formal demonstration that primitive human HSC with self-renewal and multi-lineage repopulation capacities were transduced by LV. Our findings are relevant for the design of clinical protocols that exploit this system to reach significant engraftment by genetically modified HSC in the absence of in vivo selection or strong conditioning regimens

Research needs towards a resilient community: Vulnerability reduction, infrastructural systems model, loss assessment, resilience-based design and emergency management

Most of the literature on resilience is devoted to its assessment. It seems time to move from analysis to design, to develop the tools needed to enhance resilience. Resilience enhancement, a close relative of the less fashionable risk mitigation, adds to the latter, at least in the general perception, a systemic dimension. Resilience is often paired with community, and the latter is a system. This chapter therefore discusses strategies to enhance resilience, endorses one of prevention rather than cure, and focuses in the remainder on the role played by systemic analysis, i.e. the analysis of the built environment modelled beyond a simple collection of physical assets, with due care to the associated interdependencies. Research needs are identified and include challenges in network modelling, the replacement of generic fragility curves for components, how to deal with evolving state of information

Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts

A novel homozygous change of CLCN2 (p.His590Pro) is associated with a subclinical form of leukoencephalopathy with ataxia (LKPAT)

No abstract availabl