Evidence for a higher resolution of HLA genotyping by a new NGS-based approach.

International audienceWith more than 16,000 alleles identified, the human leucocyte antigen (HLA) system is one of the most polymorphic regions of the human genome. Regarding the crucial role of HLA compatibility in transplantation and especially in Hematopoietic Stem Cell Transplantation, identification of HLA polymorphisms at a high-resolution level is of major interest. Recently, NGS technology has been proposed which appears to be simpler and more informative than the classical molecular methods such as SSP, SSOr and SBT. In the present report, a new set of NGS reagents and the appropriate associated software for sequence analysis are described. Through different studies, the performances of the system are illustrated and demonstrate that the method herein described overcomes current limitations in performing high-resolution HLA typing in clinical laboratories

Trunk Movement Analysis with FDA: Exploratory Study for the Healthy Subject

In this work, 48 acquisitions of trunk flexion-extension in healthy subjects are evaluated with the Functional Data Analysis (FDA) approach. Signals are registered with different strategies: i) assigning to the registering function two and three landmark points, and ii) aligning signals to a target profile externally imposed. The results are compared, and the normality profile for the analyzed sample is computed, revealing that the registration with three landmark points represents the best compromise between accuracy and computational burden for the current dataset. The reliability of FDA for the current purpose is investigated with the Leave-One-Out method, and the obtained results suggest the suitability of FDA for the analysis of this kind of data, although further studies should be performed increasing the original dataset in quantity and quality to extend the reliability of the normality profile to the whole healthy population

A new set of reagents and related software used for NGS based classical and non-classical HLA typing showing evidence for a greater HLA haplotype diversity

International audienceTo evaluate the HLA typing performance of a new Long-Range PCR NGS set of reagents and its dedicated software, a panel of 41 reference homozygous cell lines from the International Histocompatibility Working Group (IHWG) and a panel of 376 volunteer bone marrow donors were analyzed for classical and non-classical HLA class I and class II genes. All results, except HLA-DPB1, were obtained without any ambiguities at the 3rd field level. Based on the high resolution performance of the reagents, a number of new alleles have been described not only for classical but also for non-classical HLA class I genes, leading to a more accurate haplotype definition. Linkage disequilibrium between HLA-A and HLA-G genes has been defined at 4th field level of resolution. Moreover, for the first time, HLA-DQA2 and DQB2 polymorphisms and their linkage disequilibrium with DQB1 were described

The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform

International audienceBACKGROUND: The von Hippel-Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies. METHODS: We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array. RESULTS: We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172. CONCLUSIONS: The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.British Journal of Cancer advance online publication 2 June 2015 doi:10.1038/bjc.2015.189 www.bjcancer.co