This project represents is a continuation of series of collaborative studies performed to better characterize and understand immune deficiency. Mutations involving the genes for the common gamma chain (X-SCID) and Fas (ALPS) are being evaluated using Sanger sequencing of genomic DNA with fluorescent probes. These studies have continued to identify a number of new mutations in both diseases and these data have been entered into the NIH NHGRI web site supporting each of these two disorders. This was followed by the inclusion of mutation analysis of patients with hyper IgM syndrome directed at the genes encoding CD40L and NEMO followed by sequencing for immune deficiency associated mutations focused on host defense defects with recurrent infections involving opportunisitc intracellular organisms including genes encoding the interferon gamma receptor 1 and 2, the IL-12P40 and IL-12 receptor beta 1 genes. Finally, new additional genes have been added to the repertoire including genes encoding: AIRE, ARTEMIS, BTK, FOXP3, ICOS, IL-7Ralpha, JAK3, mu heavy chain,SAP, WASp. This initial work is now complemented by NextGen sequencing using the Ion Torrent PGM platform focused on expanding number of genes evaluated associated with primary immunodeficiencies that currently is focused on 172 known or possible primary immunodeficiency genes to screen patients refered to the NIH and on clinical research protocols with suspected primary immunodeficiency disorders. THis approach to gene mutation screeningd depends on emulsion PCR platform (Haloplex system) that has been validated in house. This togetehr with the choice of the appropriate library has been validated using deidentified patient gDNA samples with previously defined mutations linked to primary immunodeficiency disorders. To date we have identified at least a number of new genes associated with a previously uncharacterized primary immunodeficiency that have been confirmed by Sanger sequencing and functional testing. We continue to evaluate additional samples and will be moving to a more robust platform using the same principle but potentially allowing whole exome sequencing as well as targeted gene sequencing. We are in the process of preparing for publication our overall experience with the targeted gene approach (using the Ion Torrent and Haloplex technology) in screening a substantial number of patient samples. Our expereince to date suggests that this is a cost effective approach for screening referred patients with clear evidence of defects in host defense and that resequencing using the standard Sanger method under defined circumstances may not be necessary.
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